Secondary Antibodies for Immunofluorescence (IF)

Immunofluorescence (IF) is a term that describes the use of fluorophore-labeled antibodies for detecting targets of interest in biological samples. It can be applied for a broad range of research techniques, including flow cytometry, immunocytochemistry (ICC), and immunohistochemistry (IHC), as well as western blotting, enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). A main advantage of immunofluorescence over chromogenic or chemiluminescent detection is that it allows for multiplexing. Depending on the type of immunoassay being performed, immunofluorescence permits as many as 20 or more analytes to be measured simultaneously. Assays based on immunofluorescence can employ either direct or indirect detection. During direct detection, the primary antibodies are labeled with fluorophores, meaning fewer protocol steps than indirect detection. A further benefit of direct detection is that it permits using multiple antibodies from the same host species in a single experiment. Indirect detection instead combines unlabeled primary antibodies with labeled secondary antibody reagents, which provides signal amplification as a result of multiple secondary antibodies binding to each primary antibody. Importantly, indirect detection offers increased flexibility for panel design due to the broad range of fluorophore-labeled secondary antibodies that is commercially available. Another way of performing indirect immunofluorescent detection involves using a biotinylated secondary antibody in combination with a fluorophore-labeled streptavidin reagent. This represents a popular approach to boost the assay sensitivity and improve the detection of low abundance analytes, despite introducing an extra protocol step (and associated washes) into experimental workflows. When selecting secondary antibodies for immunofluorescence, it is important to match the excitation and emission maxima of each fluorophore to the lasers and detectors of the system that will be used for analysis. In addition, the chosen fluorophores should be spectrally distinct from one another, as well as from fluorescent nuclear counterstains such as 4', 6-diamidino-2-phenylindole (DAPI), Hoechst 33342, or propidium iodide (PI) in applications where these are used, to avoid spectral overlap that could lead to false positive results. Other general recommendations when using secondary antibodies for immunofluorescence include blocking with a serum that shares the same host species as the secondary antibody reagent to minimize unwanted background staining, and ensuring that the secondary antibodies are specific for the primary antibodies they are intended to detect. This is easily checked by running a secondary antibody only control (no primary antibody incubation step) to identify any non-specific secondary antibody binding.